Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems

Abstract

The PglB oligosaccharyltransferase (OTase) of Campylobacter jejuni can be functionally expressed in Escherichia coli, and its relaxed oligosaccharide substrate specificity allows the transfer of different glycans from the lipid carrier undecaprenyl pyrophosphate to an acceptor protein. To investigate the substrate specificity of PglB, we tested the transfer of a set of lipid-linked polysaccharides in E. coli and Salmonella enterica serovar Typhimurium. A hexose linked to the C-6 of the monosaccharide at the reducing end did not inhibit the transfer of the O antigen to the acceptor protein. However, PglB required an acetamido group at the C-2. A model for the mechanism of PglB involving this functional group was proposed. Previous experiments have shown that eukaryotic OTases have the same requirement, suggesting that eukaryotic and prokaryotic OTases catalyze the transfer of oligosaccharides by a conserved mechanism. Moreover, we demonstrated the functional transfer of the C. jejuni glycosylation system into S. enterica. The elucidation of the mechanism of action and the substrate specificity of PglB represents the foundation for engineering glycoproteins that will have an impact on biotechnology.

Fig. 1.

Transfer of a single O16 antigen subunit to AcrA. (A) Structure of the repetitive O16 subunit. Galf, Galactofuranose; Rha, rhamnose. (B) AcrA was purified from EVV11 (Δwzy) cells carrying the rhamnosyltransferase gene wbbL and expressing PglB (lane 1) or PglB_mut (lane 2), separated by SDS/PAGE, and visualized by Coomassie blue staining.

Fig. 2.

Glycosylation of AcrA in E. coli E69 (O9a/K30) and its derivatives CWG28 (O9a/K30⁻) and CWG44 (O9a⁻/K30). (A) Structure of different polysaccharides in E. coli E69 cells. The O9a polymannan is synthesized by the ABC transporter-dependent mechanism and is linked to the lipid A core, whereas the K30 polysaccharide is the established prototype for group 1 capsules. GlcA, glucuronic acid; Man, mannose. (B) Proteins derived from periplasmic extracts (Left) as well as Ni²⁺ affinity-purified proteins from the same extracts (Right) derived from E. coli cells expressing AcrA and PglB or PglB_mut were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected with antibodies directed against either AcrA (lanes 1–6) or Con A lectin (lanes 7–12). (C) Structure of the oligosaccharides detected by tandem MS linked to AcrA in strain CWG44 (O9a⁻/K30). For details on the MS results, see Supporting Text.

Fig. 3.

Glycosylation of AcrA in S. enterica LT2. (A) Structure of the Salmonella LT2 O-antigen subunit. Man, mannose; Rha, rhamnose; Abe, abequose. (B) Western blot analysis of S. enterica SL3749 (ΔWaaL) periplasmic extracts expressing AcrA, and the pgl locus containing either PglB_wt or PglB_mut. Antisera anti-AcrA (lanes 1 and 2) or R12 (lanes 3 and 4) were used. (C Left) Western blot analysis of S. enterica SL3749 whole-cell extracts expressing AcrA and PglB_wt or PglB_mut in the absence of the pgl locus, showing production of S. enterica LT2 Und-PP-bound O antigen. Crossreaction of the antisera with AcrA can be detected. (C Right) Western blot of purified AcrA from periplasmic extracts of the cells is shown. Only one band, corresponding to the unglycosylated protein, is detectable in the presence of PglB_wt or PglB_mut.

Fig. 4.

A mechanism to explain the necessity of the 2-acetamido group for carbohydrate transfer by PglB involves initial enzyme activation of the undecaprenol diphosphate. After cleavage of the anomeric linkage, the acetamido group acts as a neighboring group to stabilize the resulting oxonium intermediate before nucleophilic attack by the amide of an asparagine side chain. The electrophile created is reactive enough not to require deprotonation of the asparagine. The enzyme active site serves to hold the substrates in proximity and exclude water from the reactive intermediates.